In the previous stage of this project (supported by DA 13261-01 through 05), we have studied the impact of Na+ and Cl- on dopamine (DA) binding to the human DA transporter (hDAT) and the role of membrane potential along with transmembrane ion gradients;substrate-like compounds with some preference for the cocaine vs. DA binding domain on the DAT;and DAT residues involved in ion sensitivity, in particular conserved Trp and acidic residues. Now we wish to further characterize the structural features governing the interaction of cocaine-like, benztropine/GBR 12909-like, and substrate-like compounds with DAT, and further study Cl- modulation of the transporter, as follows. (1) To assess the structural feature(s) of the benztropine/GBR 12909 molecule distinguishing its binding from that of cocaine. We will study the difference between CFT/cocaine, on the one hand, and benztropine/GBR 12909, on the other hand, in interacting with DAT, using our previously developed W84L and D313N mutants, combined with structural analogs of cocaine, benztropine, and GBR 12909. The structure-activity relationships (SAR) for these inhibitors will also be assessed for their ability to alter chemical modification of DAT Cys residues and to increase the surface presence of DAT by trafficking effects. (2) To pinpoint groups of cocaine (DA) interacting with certain DAT residues by combining structural changes in cocaine (DA) with mutations in DAT. We will study the role of hydrogen bonding between carboxyl groups in cocaine (or hydroxyls in DA) and hydroxyls of tyrosine or serine in DAT, or cation-pi interaction between nitrogen and aromatics. (3) To study the SAR of substrate-derived compounds in interacting with DAT as a substrate, inhibitor, or potential cocaine antagonist;effects on DAT trafficking will also be assessed. We will focus on DA-like compounds, aminonaphthalenes with added catechol, and bivalent ligands of dimerized DA-like or naphthalene-like structures, potentially binding to DAT dimers. (4) To study the role of Cl- in DAT function and identify conserved Arg and Lys residues controlling Cl- modulation of the transporter. We will study the role of Cl- with our previously developed mutants that prefer either the outward- or inward-facing state as well as by varying concentrations of Na+ or Zn2+ that promote DAT conformational changes. The Cl-dependence of substrate binding will also be studied.